The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
Sequence reaction products must be labeled. This can be done using labeled primers, labeled nucleotides (usually radioactive dNTPs) or labeled ddNTP terminators. The use of labeled terminators has the advantage of leaving false-stops undetectable.
DNA sequence bands do not necessarily have uniform intensities. It is useful to express band intensity variability numerically. This can be done by reporting the ratio of maximum to minimum intensity of nearby bands (within a window of perhaps 40 bases)in a DNA sequence or, with normalization and correction for systematic “drift” in intensity by reporting the root mean square of band intensities (typically peak heights)(Fuller, C. W., Comments 16(3):1-8, 1989). It is advantageous to have uniformity of band intensity as sequence accuracy and read-length is improved with bands of more uniform intensity.
For accurate reading, the mobility of any given sequencing reaction product must migrate through the electrophoresis gel with a speed proportional only to its length. Products which migrate faster or slower than normal for a given length will result in sequence ambiguities or errors known as “compressions”.
Anomalous migration speed can be caused by secondary structure of the DNA and is apparently the cause of most “compression” artifacts seen in radioactive-label (and other) sequencing experiments at GC-rich regions. These can often be resolved by the use of analogs of dGTP such as 7-deaza-dGTP or dITP. Another compression-like artifact is observed when some dye-labeled ddNTPs are used for sequencing. Several examples of this can be seen in Lee, L. G., Connell, C. R., Woo, S. L., Cheng, R. D., McArdle, B. F., Fuller, C. W., Halloran, N. C., and Wilson. R. K., Nucleic Acids Res., 20:2471-2483, 1992 (see FIGS. 4g, 4h and 6h using ddCTP labeled with tetramethylbodipy and TMR or ddGTP labeled with bifluor). These compression-like artifacts are produced, even in sequences which are compression-free when sequenced radioactively or with dye-labeled primers. These artifacts can sometimes be eliminated by substituting dITP for dGTP or alpha-thio dNTPs for normal dNTPs (Lee, L. G. et al., Nucleic Acids Res., 20: 2471-2483, 1992; U.S. Pat. No. 5,187,085). Similar artifacts seen with the fluorescein dye-labeled ddNTPs sold by Applied Biosystems for dye-terminator sequencing with T7 DNA polymerase are resolved by substituting alpha-thio dNTPs for normal dNTPs (Lee, L. G. et al., Nucleic Acids Res., 20: 2471-2483, 1992; U.S. Pat. No. 5,187,085).
Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A. and Baumeister, K., Science 238:336-41 (1987) performed sequencing using terminators labeled with substituted succinyl-fluoresceins with linkers of 10 atoms in length, together with dATP, dCTP, dTTP, 7-deaza-dGTP and AMV reverse transcriptase, and a fluorescence-detecting instrument. From FIG. 6 of this paper is clear that overall band intensities varied by more than 10-fold, far more than the best available current methods with dye primers or radioactive labels.
Dideoxy NTP terminators that have the same basic structure as the Prober et al. (1987) terminators, but have four rhodamine dyes used in place of the succinyl fluoresceins and linkers of 5 atoms in length, have been used for sequencing with Taq polymerase. In order to use these terminators, dITP is used in place of dGTP or 7-deaza-dGTP to eliminate severe “compression” artifacts. This method has been practiced using cloned Taq DNA polymerase(Bergot, WO 9105060; Parker, L. T., Deng, Q, Zakeri, H., Carlson, C. Nickerson, D. A., Kwok, P. Y., Biotechniques 19(1):116-121, 1995) and with a mutant of Taq polymerase (D49G, AmpliTaq CS) lacking 5′-3′ exonuclease activity. However, band intensities vary by as much as 20-fold, limiting the accuracy and read-length possible with the method (Parker, L. T., Zakeri, H., Deng, Q., Spurgeon, S., Kwok, P. Y., Nickerson, D. A., Biotechniques 21(4):694-699, 1996).
Lee, L. G., Connell, C. R., Woo, S. L., Cheng, R. D., McArdle, B. F., Fuller, C. W., Hallorand, N. D. and Wilson, R. K., Nucleic Acids Res., 20:2471,1992)describe sequencing with a set of ddNTP terminators and T7 DNA polymerase. All have fluorescein-type dyes attached to the ddNTPs in essentially the same manner as the rhodamine terminators used for Taq sequencing. These are used with modified T7 DNA polymerase (Sequenase™ version 2.0) and alpha-thio dNTPs. The thio dNTPs are used to resolve the “compression” artifacts like dITP is used for the Taq dye-terminator methods. The results with this system are such that bands vary in intensity about 10-fold.
Wayne Barnes has published a protocol for dye-terminator sequencing with FY modified polymerases and Mn2+ (Scientech Corp. St. Louis, Mo.). Bands are more uniform with this method varying about 4.5-fold at most.
Fluorescein-12 ddNTPs that have a linker length of 12 atoms and Biotin-11 ddNTPs that have a linker length of 11 atoms are available (Dupont NEN, Wilmington, Del.). These labeled ddNTPs are described as useful in sequencing reactions.
ABI PRISM disclose dichlororhodamine dyes linked to terminators by propargyl/ethylene oxide/amino (“EO”) linkers eight atoms in length for sequencing (Rosenblum, B. B., Lee, L. G., Spurgeon, S. L., Khan, S. H., Menchen, S. M., Heiner, C. R., and Chen, S. M., Nucleic Acids Res. 25(22):4500-4504, 1997).
Cyanine dyes have been utilized in dye terminators for sequencing (Lee et al., Nucleic Acids Res., 20(10) :2471, 1992).